Molecular mechanisms of host restriction of arenaviruses and viral antagonism

The importance of cell-intrinsic or host restriction factors of the innate immune response in shaping the life cycle of arenaviruses, which pose a significant global disease burden, is poorly understood. I discovered the cell-intrinsic inhibitory activity of the zinc metalloprotease ZMPSTE24 against arenavirus entry and identified that interferon-induced transmembrane proteins (IFITMs) co-operatively enhance ZMPSTE24-mediated viral restriction. Their expression affects cellular endocytosis kinetics implying a virus invasion barrier mechanism, and that membrane structure perturbation impacts on their activity, highlighting the importance of membrane integrity for arenavirus fusion. This proposal will define the restriction mechanism of ZMPSTE24 and IFITMs and determine their role in arenavirus infection dynamics: Objective 1 will determine their effects on host membrane structure and dynamics by measuring in-cell restriction factor-imposed differences in the freedom of motion of membranes by site-directed spin labelling coupled to Electron Paramagnetic Resonance spectroscopy. Heterogeneity in cell lipid order will also be monitored by two-photon emission and confocal with spectral emission microscopy using the membrane probe, NR12S. Objective 2 will elucidate whether arenavirus matrix protein Z, which interacts with ZMPSTE24, has a direct role in antagonising ZMPSTE24 activity, informed by mutagenesis and virus budding assays; thereby highlighting an immune escape strategy. Objective 3 will reveal how ZMPSTE24 restriction impacts on the life cycle of arenaviruses: quantitative proteomics will measure viral and host protein changes at different infection stages in the presence of ZMPSTE24 and IFITMs and identify key processes modulated by these restriction factors. Altogether, this multidisciplinary approach will unravel the impact of host restriction mechanisms on arenavirus pathogenesis and open up avenues for targeted therapeutic strategies.